Acellular biologic composition and method of manufacture

ABSTRACT

A biological composition has a mixture of mechanically selected allogeneic biologic material derived from placental tissue. The mixture has non-whole cellular components including vesicular components and active and inactive components of biological activity, cell fragments, cellular excretions, cellular derivatives, and extracellular components. The mixture including non-whole cell fractions including one or more of exosomes, transcriptosomes, proteasomes, membrane rafts, lipid rafts. The mixture is compatible with biologic function.

RELATED APPLICATIONS

This application is a division of co-pending application U.S. Ser. No.14/810,003 filed on Jul. 27, 2015 entitled, “Acellular BiologicComposition And Method Of Manufacture”.

TECHNICAL FIELD

This invention is a tissue regenerative biological composition. Morespecifically, a composition at least in part formed from placentalcontents and a method of manufacture and use of said composition with anacellular mixture.

BACKGROUND OF THE INVENTION

In the area of tissue regeneration or repair, the use of stem celltherapy has been widely touted as essential to sustaining regenerativeinertia in tissue repair and guiding sufficient metabolic inertia tosupplement matrix deposition and organ consolidation.

Often, these inventions describe isolating the stem cells, purifying andculturally expanding mesenchymal stem cells. In U.S. Pat. No. 5,837,539,entitled “Monoclonal Antibodies For Human Mesenchymal Stem Cells”,Arnold Caplan et al. reported that the cells are preferably culturallyexpanded, but suggest it is possible to use the stem cells withoutculture expansion. Caplan also describes a way to isolate stem cells.Although that work was developed from bone marrow isolation, subsequentobservations have shown that pluripotent cells are present and can beisolated from nearly all tissues. Particular attention has been attendedto isolation from adipose tissue, but the abundance and availability ofplacental tissue has also garnered a huge interest because of its readysupply.

A major technological hurdle to producing a safe allogeneic compositionwith viable cells has been the need to approach a fraction of riskapproaching zero by removing all antigenic properties that lead toinflammation factors in a separation to yield only a certain stromalcell type. This has proven both difficult and degrading as the quantityof viable cells that can be effectively harvested is greatly diminishedby the process itself.

The present invention has yielded a biological composition that is safeand achieves regenerative support and does so in a method that allowsthe resultant mixture to be recovered from placental tissues wherein themixture unexpectedly exhibits evidence of regenerative capacitysustaining a legacy, or memory of the lineages from where the acellularbiological composition had been associated. This retained ability, orregenerative resonance to support the emergence of new tissue formsincluding bone and other tissues was independent of mesenchymal cells.

These and other benefits of the present invention and the method ofpreparing it are described hereinafter.

SUMMARY OF THE INVENTION

A biological composition has a mixture of mechanically selectedallogeneic biologic material derived from placental tissues. The mixturehas non-whole cellular components including vesicular components andactive and inactive components of biological activity, cell fragments,cellular excretions, cellular derivatives, and extracellular matrixcomponents. The mixture including non-whole cell fractions including oneor more of exosomes, transcriptosomes, proteasomes, membrane rafts,lipid rafts. The mixture is compatible with biologic function.

The mixture of mechanically selected material is derived from placentaltissues. The biological composition preferably has placental particlesincluding derivatives of the amnion, chorion, epithelial layer, and caninclude non-whole cellular fragments of the umbilical cord and fetal andmaternal vessels including Wharton Jelly. Mesenchymal cells derived fromamniotic tissue can be added to the mixture derived from placentaltissues.

The combination of non-whole cell components with a select number ofnon-whole cell fractions sustains pluripotency in the cells. In apreferred embodiment, the biological composition is predisposed todemonstrate or support elaboration of active volume or spatial geometryconsistent in morphology with host specific tissue and organ geometry.The biological composition extends regenerative significance thatcompliments or mimics tissue complexity. The mixture is treated in aprotectant or cryoprotectant prior to preservation or cryopreservationor freeze drying. The composition can be maintained at ambienttemperature prior to freeze drying. The protectant or cryoprotectantcreates a physical or electrical or chemical gradient or combinationthereof for tissue regeneration. The gradient can have a physicalcharacteristic of modulus or topography, such as charge density, fieldshape or cryo or chemotaxis tendencies. The gradient can have a chemicalcharacteristic of spatially changing compositions of density or speciesof functional molecules, wherein the molecules can offer a fixedcatalytic function as a co-factor. Also, the gradient can have anelectrical characteristic of charge based or pH based or electronaffinities that confer metastability in biologic potential.

The placental tissue mixture which is donor derived from a Caesareansection has separation-enhanced non-whole cell fraction vitalityincluding one or more of the following: separating the fractions fromcells heightens their vital potency, reversing “arrest” of donor tissuefollowing birth process, responsive molecular coupling, matrix quest inneutralizing inflammation or satience by balancing stimulus for repair.The protectant or cryoprotectant is a polyampholyte. The regenerativeresonance occurs in the presence or absence of a refractory response.When using a cryoprotectant, the cryopreservation occurs at atemperature that is sub-freezing wherein the cryopreservationtemperature is from 0 degrees C. to −200 degrees C. The protection mayalso be achieved by non-cryogenic means.

The biological composition's non-whole cellular component also caninclude organelle fragments and the active and inactive components ofbiological activity which can also include extants of the humanmetabolome.

A method of making a biological composition of the present invention hasthe steps of: collecting, recovering and processing placental tissuefrom a donor; mechanically separating the cellular or non-cellularcomponents or a combination thereof of placental tissue from surgicalC-section; concentrating by centrifugation and filtering; separation bydensity gradient centrifugation; collecting non-cellular fractions ornon-cellular components or a combination thereof of predetermineddensity; washing the non-whole cellular fractions or non-cellularcomponents or a combination thereof to create the mixture; quantifyingconcentrations of non-cellular fractions components at a non-zeroentity; suspending to a predetermined concentration in a polyampholytecryoprotectant; freezing the mixture at a predetermined controlled rate;and packaging a placental-derived blend having particles in the sizerange of 100 to 300 μm of chorion, amnion, umbilical cord matrix,Wharton's Jelly, within the mixture or separate, or in combination.Novel acellular materials including exosomes have been shown to rangefrom 30-120 nm as endocytic membrane-derived vesicles, and have beenshown in several reviews that participate in cell-to-cell communicationand protein and RNA delivery. These particle size ranges can vary higheror lower depending on the application. At the time of use, the mixtureis thawed by immersion in a warm water bath for 2-3 minutes at 37degrees C., or held in hand until liquid It is diluted in saline withoutspinning; and then the diluted mixture, with or without the morselizedmatrix materials being intermixed, can be implanted by packing,injection, scaffolding or any other suitable means into a patient.

Definitions

DNase—deoxyribonuclease is any enzyme that catalyzes the hydrolyticcleavage of phosphodiester linkages in the DNA backbone, thus degradingDNA.

DMEM, DMEM/LG—Dulbecco's Modified Eagle Medium, low glucose. Sterile,with: Low Glucose (1 g/L), Sodium Pyruvate; without: L-glutamine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

DPBS—Dulbecco's Phosphate Buffered Saline.

Cryopreserved—Tissue frozen with the addition of, or in a solutioncontaining, a cryoprotectant agent such as glycerol, ordimethylsulfoxide, or carboxylated poly-1-lysine.

Freeze Dried/Lyophilized—Tissue dehydrated for storage by conversion ofthe water content of frozen tissue to a gaseous state under vacuum thatextracts moisture.

Normal Saline—0.9% Sodium Chloride Solution.

Packing Media—Media used during initial processing and storage of theprocessed tissue prior to decellularization.

PBS—Phosphate Buffered Saline.

Processing Media—Media used during bone decellularization that maycontain DMEM/Low Glucose no phenol red, Human Serum Albumin, Heparin,Gentamicin and DNAse.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing/photographexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

The invention will be described by way of example and with reference tothe accompanying drawings in which:

FIG. 1 Depiction of full fetal membranes from placental material,including both amnion and chorion layers from a donor placenta.

FIG. 2 Photo-micrograph depicting both the amnion and chorion layers offetal membrane.

FIG. 3 shows a photomicrograph of the amnion separated from the chorionlayer.

FIG. 4 shows umbilical cord tissue reduced to its membrane and scrapedclean of connective tissue.

FIG. 5 is a photograph showing plain amniotic fluid after filtration.

FIG. 6 shows a photograph of the placental material; e.g. placenta,chorion, amniotic membrane, umbilical cord or a mixture of these afterbeing ground and immersed in media.

FIG. 7 shows a pair of views of non-whole cell material that can bederived from supernatant of placental material recovery; the resultantmaterial after centrifuging showing the non-whole cell fractioninterface layer, including exosomes.

DETAILED DESCRIPTION OF THE INVENTION

With reference to the present invention which is a tissue regenerativebiological composition made from placental material includingderivatives of amnion, chorion, umbilical cord, Wharton's Jelly, it isbelieved best understood by the methods used to process and recover thebiological composition, as illustrated in the FIGS. 1-7.

The first steps are to collect, recover and process placental materialsfrom a C-section donor of a live birth. To do this, the placenta isremoved and collected aseptically following the birth and the resultantmaterial is covered by cold media. This also can include recovery ofamniotic fluid as shown in FIG. 3 and the cleaned and scraped umbilicalcord tissue shown in FIG. 4.

After each subsequent sieving of the placental material, exosomes canpotentially be recovered from the cold media, but also from incubationof micronized tissue with media as well as from the amniotic fluid. Thedecanted fluid, containing the mixture with whole cells is collected andput into a collection jar. When the next three cycles are complete andthe decanted fluid is all placed in the collection jar commingling thefluids to form a decanted fluid. Then the centrifugation of the combineddecanted fluid occurs by placing the fluid in a number of 250-ml conicaltubes using a 100-ml pipette. The centrifuge is programmed to 280×g for10 minutes at room temperature, preferably about 20 degrees C., orapproximately at normal room temperature. The fluid is passed through ablood filter to further remove any bone or spicules or clumps from thesuspended cells. This completes the step of centrifuging and filtering.At this point, as shown in FIG. 5, the mixture including whole cells hasbeen separated from the soft tissue and the remaining macerated andfibrous tissue is discarded.

Typically, non-whole cell fragments, or membrane components have adiameter of 40-100 nm and can be separated within a density of 1.13-1.19g/mL in a sucrose solution, and can be sedimented by centrifugation at100,000 g. In fact, these fragments, or cell fractions, ormicrovesicles, have been collectively referred to as exosomes. Rangingin size from 20-1000 nm in diameter, they have been similarly referredto as nanoparticles, microparticles, shedding microvesicles, apoptoticblebs, and human endogenous retroviral particles. There are few firmcriteria distinguishing one type of microvesicle from the other.

Following removal of the cell fraction, the supernatant is furtherfiltered through 0.45 and 0.2 μm filters. Exosomes are further collectedand separated within the suspension in multiple centrifugation stepswith increasing centrifugal strength to sequentially pellet cells (300g), microvesicles (10,000 g) and ultimately exosomes (100,000 g). Cellsare deliberately removed to achieve the non-whole cell fragments andmicrovesicles.

Subsequent separation using density gradient-based isolation, usingsucrose or commercially available prep can be applied to obtain morepure exosome preparations. Recent reports encouraging the use ofiodixanol-based gradients for improved separation of exosomes fromviruses and small apoptotic bodies are considerations left open to beadopted or adapted in refinement. Differing from sucrose, iodixanolforms iso-osmotic solutions at all densities, thus better preserving thesize of the vesicles in the gradient, and both technologies areavailable to best isolation technology. In addition to these traditionalisolation techniques, easy-to-use precipitation solutions, such asEXOQUICK™ and TOTAL EXOSOME ISOLATION™ (TEI), that have beencommercialized reduce the need for expensive equipment or technicalknow-how. Although their mode-of-action has not been disclosed orvalidated, these kits are commonly used.

Once the mixture is completed, the method can include additional steps.This leads to the use of a material composition, blends of materialsshown in FIG. 6, preferably from the same donor.

When the mixture is prepared, it can have whole cells or even no wholecells, but will have the mechanically selected non-whole cellularcomponents including vesicular components and active and inactivecomponents of biological activity, cell fragments, cellular excretions,cellular derivatives, and extracellular components from the placentaltissues, as shown in FIG. 7.

In one embodiment, the composition includes the whole cells in themixture. In that embodiment, it is possible to provide bone particleswith the mixture either in the mixture or separately to be combined atthe time of use.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed, which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A method of making a biological compositioncomprises the steps of: collecting, recovering and processing placentaltissue from a donor; mechanically separating cellular and non-cellularcomponents or combinations thereof of placental tissue; concentrating bycentrifugation and filtering to form a suspension of the cellular andnon-cellular components; separating by density gradient centrifugationto form a cell pellet of the cellular components and a supernatant ofthe non-cellular components wherein the cellular components in the cellpellet are separated from the supernatant and removed and wherein thesupernatant is further consisting of filtering through a 0.45 micronfilter; collecting non-cellular fractions or non-cellular components orcombinations thereof of predetermined density by increasingcentrifugation strength; washing the non-cellular fractions ornon-cellular components or combinations thereof; quantifying thenon-cellular fractions or non-cellular components or combinationsthereof separated by density gradient concentration is greater thanzero; suspending the non-cellular fractions or non-cellular componentsor combinations thereof to a concentration with a blend of collected,recovered and processed placental tissue and bone particles to create amixture in a cryoprotectant consisting of a polyampholyte cryoprotectantto create a biological composition, wherein the bone particles having asize range of 100 to 300 μm of demineralized cortical bone, mineralizedcortical bone and mineralized cancellous bone intermixed with thenon-cellular fractions or non-cellular components; and freezing thebiological composition at a controlled rate.
 2. The method of preparingthe biological composition for use made according to the method of claim1 by the steps of: thawing the biological composition; diluting thethawed biological composition in saline without spinning; and implantingthe diluted biological composition with the blend of additional tissuebeing intermixed by packing, injection or any other suitable means intoa patient.
 3. The method of preparing the biological composition for usemade according to the method of claim 2 wherein the step of thawing thebiological composition occurs at a temperature of 37 degrees C. for 2 to3 minutes in a warm water bath.
 4. The method of making the biologicalcomposition of claim 1 further consists of the step of adding a quantityof whole cells to the non-cellular fractions or non-cellular componentsor combinations to a concentration with the blend of collected,recovered and processed placental tissue and bone particles in thepolyampholyte cryoprotectant to create the biological composition.
 5. Amethod of making a biological composition comprises the steps of:mechanically separating cellular and non-cellular components orcombinations thereof of placental tissue; concentrating bycentrifugation and filtering to form a suspension of the cellular andnon-cellular components; separating by density gradient centrifugationto form a cell pellet of the cellular components and a supernatant ofthe non-cellular components wherein the cellular components in the cellpellet are separated from the supernatant and removed and wherein thesupernatant is further consisting of filtering through a 0.45 micronfilter; collecting non-cellular fractions or non-cellular components orcombinations thereof of predetermined density by increasingcentrifugation strength; washing the non-cellular fractions ornon-cellular components or combinations thereof; quantifying thenon-cellular fractions or non-cellular components or combinationsthereof separated by density gradient concentration is greater thanzero; suspending the non-cellular fractions or non-cellular componentsor combinations thereof to a concentration with a blend of collected andprocessed placental tissue to create a mixture in a cryoprotectantconsisting of a polyampholyte cryoprotectant to form a biologicalcomposition; and freezing the biological composition at a controlledrate.
 6. The method of preparing the biological composition for use madeaccording to the method of claim 5 by the steps of: thawing thebiological composition; diluting the thawed biological composition insaline without spinning; and implanting the diluted biologicalcomposition by packing, injection or any other suitable means into apatient.
 7. The method of preparing the biological composition for usemade according to the method of claim 6 wherein the step of thawing thebiological composition occurs at a temperature of 37 degrees C. for 2 to3 minutes in a warm water bath.
 8. The method of making the biologicalcomposition of claim 5 further consists of the step of adding a quantityof whole cells to the non-cellular fractions or non-cellular componentsor combinations to a concentration with the blend of collected,recovered and processed placental tissue in the polyampholytecryoprotectant to create the biological composition.
 9. A method ofmaking a biological composition comprises the steps of: collecting,recovering and processing placental tissue from a donor; mechanicallyseparating cellular and non-cellular components or combinations thereofof placental tissue; concentrating by centrifugation and filtering toform a suspension of the cellular and non-cellular components;separating by density gradient centrifugation to form a cell pellet ofthe cellular components and a supernatant of the non-cellular componentswherein the cellular components in the cell pellet are separated fromthe supernatant and removed and wherein the supernatant is furtherconsisting of filtering through a 0.45 micron filter; collectingnon-cellular fractions or non-cellular components or combinationsthereof of predetermined density by increasing centrifugation strength;washing the non-cellular fractions or non-cellular components orcombinations thereof; quantifying the non-cellular fractions ornon-cellular components or combinations thereof separated by densitygradient concentration is greater than zero; suspending the non-cellularfractions or non-cellular components or combinations thereof to aconcentration with a blend of collected, recovered and processedplacental tissue and bone particles to create a mixture in acryoprotectant consisting of a polyampholyte cryoprotectant to create abiological composition, wherein the bone particles having a size rangeof 100 to 300 μm of demineralized cortical bone, mineralized corticalbone and mineralized cancellous bone intermixed with the non-cellularfractions or non-cellular components; and freeze-drying the biologicalcomposition.
 10. The method of preparing the biological composition foruse of claim 9 further consists of the steps of: diluting the biologicalcomposition in saline without spinning; and implanting the dilutedbiological composition with the blend of additional tissue beingintermixed by packing, injection or any other suitable means into apatient.
 11. The method of making the biological composition of claim 9further consists of the step of adding a quantity of whole cells to thenon-cellular fractions or non-cellular components or combinations to aconcentration with the blend of collected, recovered and processedplacental tissue and bone particles in the polyampholyte cryoprotectantto create the biological composition.
 12. A method of making abiological composition comprises the steps of: mechanically separatingcellular and non-cellular components or combinations thereof ofplacental tissue; concentrating by centrifugation and filtering to forma suspension of the cellular and non-cellular components; separating bydensity gradient centrifugation to form a cell pellet of the cellularcomponents and a supernatant of the non-cellular components wherein thecellular components in the cell pellet are separated from thesupernatant and removed and wherein the supernatant is furtherconsisting of filtering through a 0.45 micron filter; collectingnon-cellular fractions or non-cellular components or combinationsthereof of predetermined density by increasing centrifugation strength;washing the non-cellular fractions or non-cellular components orcombinations thereof; quantifying the non-cellular fractions ornon-cellular components or combinations thereof separated by densitygradient concentration is greater than zero; suspending the non-cellularfractions or non-cellular components or combinations thereof to aconcentration with a blend of collected and processed placental tissueto create a mixture in a cryoprotectant consisting of a polyampholytecryoprotectant to form a biological composition; and freeze-drying thebiological composition.
 13. The method of preparing the biologicalcomposition for use of claim 12 further consists of the steps of:diluting the biological composition in saline without spinning; andimplanting the diluted biological composition with the blend ofadditional tissue being intermixed by packing, injection or any othersuitable means into a patient.
 14. The method of making the biologicalcomposition of claim 12 further consists of the step of adding aquantity of whole cells to the non-cellular fractions or non-cellularcomponents or combinations to a concentration with the blend ofcollected, recovered and processed placental tissue in the polyampholytecryoprotectant to create the biological composition.